Downregulation of HbFPS1 affects rubber biosynthesis of Hevea brasiliensis suffering from tapping panel dryness.

Tapping panel dryness (TPD) is a century-old problem that has plagued the natural rubber production of Hevea brasiliensis. TPD may result from self-protective mechanisms of H. brasiliensis in response to stresses such as excessive hormone stimulation and mechanical wounding (bark tapping). It has been hypothesized that TPD impairs rubber biosynthesis; however, the underlying mechanisms remain poorly understood. In the present study, we firstly verified that TPD-affected rubber trees exhibited lower rubber biosynthesis activity and greater rubber molecular weight compared to healthy rubber trees. We then demonstrated that HbFPS1, a key gene of rubber biosynthesis, and its expression products were downregulated in the latex of TPD-affected rubber trees, as revealed by transcriptome sequencing and iTRAQ-based proteome analysis. We further discovered that the farnesyl diphosphate synthase HbFPS1 could be recruited to small rubber particles by HbSRPP1 through protein-protein interactions to catalyze farnesyl diphosphate (FPP) synthesis and facilitate rubber biosynthesis initiation. FPP content in the latex of TPD-affected rubber trees was significantly decreased with the downregulation of HbFPS1, ultimately resulting in abnormal development of rubber particles, decreased rubber biosynthesis activity, and increased rubber molecular weight. Upstream regulator assays indicated that a novel regulator, MYB2-like, may be an important regulator of downregulation of HbFPS1 in the latex of TPD-affected rubber trees. Our findings not only provide new directions for studying the molecular events involved in rubber biosynthesis and TPD syndrome and contribute to rubber management strategies, but also broaden our knowledge of plant isoprenoid metabolism and its regulatory networks.

Increased Expression of Mevalonate Pathway-Related Enzymes in Angiotensin II-Induced Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is characterized by permanent luminal expansion and a high mortality rate due to aortic rupture. Despite the identification of abnormalities in the mevalonate pathway (MVA) in many diseases, including cardiovascular diseases, the potential impact of this pathway on AAA remains unclear. This study aims to investigate whether the expression of the MVA-related enzyme is altered during the progression of angiotensin II (Ang II) -induced AAA.Ang II 28D and Ang II 5D groups were continuously perfused with Ang II for 28 days and 5 days, respectively, and the Sham group was perfused with saline. The general and remodeling characteristics of AAA were determined by biochemical and histological analysis. Alteration of MVA-related enzyme expressions was revealed by western blot and single-cell RNA sequencing (scRNA-seq).The continuous Ang II infusion for 28 days showed significant aorta expansion and arterial remodeling. Although the arterial diameter slightly increased, the aneurysm formation was not found in Ang II induction for 5 days. MVA-related enzyme expression and activation of small GTP-binding proteins were significantly increased after Ang II-induced. As verified by scRNA-seq, the key enzyme gene expression was also higher in Ang II 28D. Similarly, it was detected that the expression levels of the above enzymes and the activity of small G proteins were elevated in the early stage of AAA as induced by Ang II infusion for 5 days.Continuous Ang II infusion-induced abdominal aortic expansion and arterial remodeling were accompanied by altered expression of key enzymes in the MVA.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Preparation and self-cleavage of fusion soluble farnesyl diphosphate synthase in E. coli.

Farnesyl diphosphate synthase (FPPS) is a crucial protein in terpenoid production. However, its industrial application is limited owing to its low solubility in Escherichia coli. In this study, we focused on ispA encoding FPPS and designed a fusion expression system to reduce inclusion body (IB) formation. Among the chosen fusion tags, the GB1-domain (GB1) exhibited the highest ability to solubilize the recombinant protein. Increased rare tRNA abundance not only improved the GB1-FPPS yield but also increased its soluble level. A "one-step" method for the acquisition of soluble FPPS was also considered. By combining GB1-FPPS expression and Tobacco Etch Virus protease (TEVp) cleavage in vivo, a controllable GB1-FPPS "self-cleavage" system was constructed. Overall, this study provides an efficient approach for obtaining soluble forms of FPPS, which show great potential for use in the soluble expression of other homologous diphosphate synthase.

Anti-Trypanosoma cruzi Potential of Vestitol Isolated from Lyophilized Red Propolis.

Chagas disease (CD) is a worldwide public health problem, and the drugs available for its treatment have severe limitations. Red propolis is a natural extract known for its high content of phenolic compounds and for having activity against T. cruzi. The aim of this study was to investigate the trypanocidal potential of red propolis to isolate, identify, and indicate the mode of action of the bioactive compounds. The results revealed that the total phenolic content was 15.4 mg GAE/g, and flavonoids were 7.2 mg QE/g. The extract was fractionated through liquid-liquid partitioning, and the trypanocidal potential of the samples was evaluated using the epimastigote forms of the Y strain of T. cruzi. In this process, one compound was characterized by MS, 1H, and 13C NMR and identified as vestitol. Cytotoxicity was evaluated employing MRC-5 fibroblasts and H9C2 cardiomyocytes, showing cytotoxic concentrations above 15.62 µg/mL and 31.25 µg/mL, respectively. In silico analyses were applied, and the data suggested that the substance had a membrane-permeation-enhancing effect, which was confirmed through an in vitro assay. Finally, a molecular docking analysis revealed a higher affinity of vestitol with farnesyl diphosphate synthase (FPPS). The identified isoflavan appears to be a promising lead compound for further development to treat Chagas disease.

Farnesyl diphosphate synthase regulated endothelial proliferation and autophagy during rat pulmonary arterial hypertension induced by monocrotaline.

BACKGROUND: The proliferation ability and autophagy level of pulmonary artery endothelial cells (PAECs) play an important role in promoting the development of pulmonary artery hypertension (PAH), and there is still no effective treatment for PAH. Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. The intermediate metabolites of this pathway are closely related to the activity of autophagy-associated small G proteins, including Ras-related C3 botulinum toxin substrate 1 (Rac1). Studies have shown that the mevalonate pathway affects the activation levels of different small G proteins, autophagy signaling pathways, vascular endothelial function, and so on. However, the exact relationship between them is still unclear in PAH. METHOD: In vitro, western blotting and mRFP-GFP-LC3 puncta formation assays were used to observe the expression of FDPS and the level of autophagy in PAECs treated with monocrotaline pyrrole (MCTP). In addition, cell proliferation and migration assays were used to assess the effect of FDPS on endothelial function, and Rac1 activity assays were used to evaluate the effect of Rac1 activation on PAEC autophagy via the PI3K/AKT/mTOR signaling pathway. In vivo, the right heart catheterization method, hematoxylin and eosin (H&E) staining and western blotting were used to determine the effect of FDPS on PAEC autophagy and monocrotaline (MCT)-induced PAH. RESULTS: We show that the expression of FDPS is increased in the PAH module in vitro and in vivo, concomitant with the induction of autophagy and the activation of Rac1. Our data demonstrate that inhibition of FDPS ameliorates endothelial function and decreases MCT-induced autophagy levels. Mechanistically, we found that FDPS promotes autophagy, Rac1 activity and endothelial disfunction through the PI3K/AKT/mTOR signaling pathway. CONCLUSION: Our study suggests that FDPS contributes to active small G protein-induced autophagy during MCT-induced PAH, which may serve as a potential therapeutic target against PAH.

Altering potato isoprenoid metabolism increases biomass and induces early flowering.

Isoprenoids constitute the largest class of plant natural products and have diverse biological functions including in plant growth and development. In potato (Solanum tuberosum), the regulatory mechanism underlying the biosynthesis of isoprenoids through the mevalonate pathway is unclear. We assessed the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) homologs in potato development and in the metabolic regulation of isoprenoid biosynthesis by generating transgenic lines with down-regulated expression (RNAi-hmgr) or overexpression (OE) of one (StHMGR1 or StHMGR3) or two genes, HMGR and farnesyl diphosphate synthase (FPS; StHMGR1/StFPS1 or StHMGR3/StFPS1). Levels of sterols, steroidal glycoalkaloids (SGAs), and plastidial isoprenoids were elevated in the OE-HMGR1, OE-HMGR1/FPS1, and OE-HMGR3/FPS1 lines, and these plants exhibited early flowering, increased stem height, increased biomass, and increased total tuber weight. However, OE-HMGR3 lines showed dwarfism and had the highest sterol amounts, but without an increase in SGA levels, supporting a rate-limiting role for HMGR3 in the accumulation of sterols. Potato RNAi-hmgr lines showed inhibited growth and reduced cytosolic isoprenoid levels. We also determined the relative importance of transcriptional control at regulatory points of isoprenoid precursor biosynthesis by assessing gene-metabolite correlations. These findings provide novel insights into specific end-products of the sterol pathway and could be important for crop yield and bioenergy crops.

[Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens].

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize ß-macrocarpene synthase) into BL21 competent cells. The results showed that the content of ß-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum.

BACKGROUND: Ganoderic acids (GAs), derived from the medicinal mushroom Ganoderma lucidum, possess anticancer and other important pharmacological activities. To improve production of GAs, a homologous farnesyl diphosphate synthase (FPS) gene was overexpressed in G. lucidum. Moreover, the influence of FPS gene overexpression on GA production was investigated by developing the corresponding mathematical models. RESULTS: The maximum levels of total GAs and individual GAs (GA-T, GA-S, and GA-Me) in the transgenic strain were 2.76 mg/100 mg dry weight (DW), 41 ± 2, 21 ± 5, and 28 ± 1 µg/100 mg DW, respectively, which were increased by 2.28-, 2.27-, 2.62-, and 2.80-folds compared with those in the control. Transcription levels of squalene synthase (SQS) and lanosterol synthase (LS) genes during GA biosynthesis were upregulated by 2.28- and 1.73-folds, respectively, in the transgenic G. lucidum. In addition, the developed unstructured models had a satisfactory fit for the process of GA production in submerged cultures of G. lucidum. Analysis of the kinetic process showed that FPS gene overexpression had a stronger positive impact on GA production compared with its influence on cell growth. Also, FPS gene overexpression led to a higher non-growth-associated-constant ß (1.151) over the growth-associated-constant α (0.026) in the developed models. CONCLUSIONS: FPS gene overexpression is an effective strategy to improve the production of GAs in G. lucidum. The developed mathematical models are useful for developing a better GA production process in future large-scale bioreactors.

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation.

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.

Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae.

Farnesyl diphosphate synthase (FPPS) catalyzes the formation of FPP, providing the precursor for the biosynthesis of (E)-ß-farnesene (EßF) in plants, but it is unknown if FPPS supplies the precursor for the biosynthesis of EßF, the major component of aphid alarm pheromone, though our previous studies support the hypothesis that EßF is synthesized by the aphid itself. Here, we used two cohorts of the green peach aphid Myzus persicae separately, reared on pepper plant and artificial diet to test the correlations among droplet emission, EßF quantity, and FPPS gene expression. It was found that the proportion of aphids emitting cornicle droplets and the quantity of EßF per milligram of aphid were both significantly different between the two cohorts, which were positively correlated with the expression of the two FPPS genes ( MpFPPS1/ 2) in M. persicae. These results were further confirmed by RNAi-mediated knockdown of MpFPPS1/ 2. Specifically, knockdown of MpFPPS1/ 2 imposed no significant cost on the survival of aphid but remarkably increased the number of offspring per aphid; most importantly, knockdown of MpFPPS1/ 2 significantly reduced the proportion of aphids emitting droplets and the quantity of EßF calculated as per the weight of aphid. Our results suggest that both FPPS genes are involved in the production of EßF in M. persicae and cornicle droplet emission is closely associated with the EßF release in the aphid.

Production of alarm pheromone starts at embryo stage and is modulated by rearing conditions and farnesyl diphosphate synthase genes in the bird cherry-oat aphid Rhopalosiphum padi.

The major component of aphid alarm pheromone is (E)-ß-farnesene (EßF), but the molecular mechanisms of EßF synthesis are poorly understood. Here we established a biological model to study the modulation of EßF synthesis in the bird cherry-oat aphid Rhopalosiphum padi by using quantitative polymerase chain reaction, gas chromatography/mass spectrometry and RNA interference. Our results showed that the rearing conditions significantly affected the weight of adult and modulated EßF synthesis in a transgenerational manner. Specifically, the quantity of EßF per milligram of aphid was significantly reduced in the individually reared adult or 1st-instar nymphs derived from 1-day-old adult reared individually, but EßF in the nymph derived from 2-day-old adult that experienced collective conditions returned to normal. Further study revealed that the production of EßF started in embryo and was extended to early nymphal stage, which was modulated by farnesyl diphosphate synthase genes (RpFPPS1 and RpFPPS2) and rearing conditions. Knockdown of RpFPPS1 and RpFPPS2 confirmed the role played by FPPS in the biosynthesis of aphid alarm pheromone. Our results suggested that the production of EßF starts at the embryo stage and is modulated by FPPS and rearing conditions in R. padi, which sheds lights on the modulatory mechanisms of EßF in the aphid.

Spatiotemporal expression profiling of the farnesyl diphosphate synthase genes in aphids and analysis of their associations with the biosynthesis of alarm pheromone.

The alarm behavior plays a key role in the ecology of aphids, but the site and molecular mechanism for the biosynthesis of aphid alarm pheromone are largely unknown. Farnesyl diphosphate synthase (FPPS) catalyzes the synthesis of FPP, providing the precursor for the alarm pheromone (E)-ß-farnesene (EßF), and we speculate that FPPS is closely associated with the biosynthetic pathway of EßF. We firstly analyzed the spatiotemporal expression of FPPS genes by using quantitative reverse transcription-polymerase chain reaction, showing that they were expressed uninterruptedly from the embryonic stage to adult stage, with an obvious increasing trend from embryo to 4th-instar in the green peach aphid Myzus persicae, but FPPS1 had an overall significantly higher expression level than FPPS2; both FPPS1 and FPPS2 exhibited the highest expression in the cornicle area. This expression pattern was verified in Acyrthosiphon pisum, suggesting that FPPS1 may play a more important role in aphids and the cornicle area is most likely the site for EßF biosynthesis. We thus conducted a quantitative measurement of EßF in M. persicae by gas chromatography-mass spectrometry. The data obtained were used to perform an association analysis with the expression data, revealing that the content of EßF per aphid was significantly correlated with the mean weight per aphid (r = 0.8534, P = 0.0307) and the expression level of FPPS1 (r = 0.9134, P = 0.0109), but not with that of FPPS2 (r = 0.4113, P = 0.4179); the concentration of EßF per milligram of aphid was not correlated with the mean weight per aphid or the expression level of FPPS genes. These data suggest that FPPS1 may play a key role in the biosynthesis of aphid alarm pheromone.

Expansion of human γδ T cells for adoptive immunotherapy using a bisphosphonate prodrug.

Cancer immunotherapy with human γδ T cells expressing Vγ2Vδ2 T cell receptor (also termed Vγ9Vδ2) has shown promise because of their ability to recognize and kill most types of tumors in a major histocombatibility complex (MHC) -unrestricted fashion that is independent of the number of tumor mutations. In clinical trials, adoptive transfer of Vγ2Vδ2 T cells has been shown to be safe and does not require preconditioning. In this report, we describe a method for preparing highly enriched human Vγ2Vδ2 T cells using the bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA). PTA stimulated the expansion of Vγ2Vδ2 cells to purities up to 99%. These levels were consistently higher than those observed after expansion with zoledronic acid, the most commonly used stimulator for clinical trials. Cell numbers also averaged more than those obtained with zoledronic acid and the expanded Vγ2Vδ2 cells exhibited high cytotoxicity against tumor cells. The high purity of Vγ2Vδ2 cells expanded by PTA increased engraftment success in immunodeficient NOG mice. Even low levels of contaminating αß T cells resulted in some mice with circulating human αß T cells rather than Vγ2Vδ2 cells. Vγ2Vδ2 cells from engrafted NOG mice upregulated CD25 and secreted tumor necrosis factor-α and interferon-γ in response to PTA-treated tumor cells. Thus, PTA expands Vγ2Vδ2 T cells to higher purity than zoledronic acid. The high purities allow the successful engraftment of immunodeficient mice without further purification and may speed up the development of allogeneic Vγ2Vδ2 T cell therapies derived from HLA-matched normal donors for patients with poor autologous Vγ2Vδ2 T cell responses.

The terpenoid backbone biosynthesis pathway directly affects the biosynthesis of alarm pheromone in the aphid.

The terpenoid backbone biosynthesis pathway is responsible for the synthesis of different backbones for terpenoids; (E)-ß-farnesene (EßF), a sesquiterpene, is the major component of aphid alarm pheromone. Our previous studies eliminated the possibility of host plants and endosymbionts as the sources of EßF, and we thus speculate that the terpenoid pathway might affect the biosynthesis of EßF in aphids. First, the transcriptional responses of four genes encoding farnesyl diphosphate synthase (FPPS), geranylgeranyl diphosphate synthase (GGPPS) and decaprenyl diphosphate synthase in the cotton aphid Aphis gossypii to simulated stimulation were analysed using quantitative real-time PCR, showing an immediate decrease in the transcript abundances of the four genes. Next, RNA-interference-mediated gene knockdown was performed, indicating that fpps knockdown caused a significant cost in terms of body size and fecundity. Finally, an association analysis of gene knockdown with the amount of EßF was conducted, revealing that the concentration of EßF per milligram of aphid was drastically decreased in response to fpps knockdown, whereas ggpps knockdown significantly raised the concentration of EßF. Our data support a peculiar mode of biosynthesis and storage of the aphid alarm pheromone that relies directly on the terpenoid backbone biosynthesis pathway in the aphid.

High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains.

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.

In Vitro and In Vivo Activities of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites.

We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound (compound 22; 1-[(n-decylsulfonyl)ethyl]-1,1-bisphosphonic acid) is a sulfone-containing compound, which had a 50% effective concentration (EC50) of 0.11 ± 0.02 µM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. Compound 22 also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS), but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested compound 22 against two other apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 µM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 µM), the agent of cryptosporidiosis. Our results suggest that compound 22 is an excellent novel compound that could lead to the development of potent agents against apicomplexan parasites.

Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites.

Based on crystallographic data of the complexes 2-alkyl(amino)ethyl-1,1-bisphosphonates-Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(amino)ethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

Fermentative production and direct extraction of (-)-α-bisabolol in metabolically engineered Escherichia coli.

BACKGROUND: (-)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (-)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock. RESULTS: Here, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%). CONCLUSIONS: The engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale.

Characterization of a farnesyl diphosphate synthase gene from Penicillium brevicompactum MUCL 19011.

OBJECTIVES: Farnesyl diphosphate synthase is a critical enzyme in the isoprenoids biosynthesis pathway responsible for ergosterol and secondary metabolites biosynthesis in fungi. RESULTS: Characterization of fds from Penicillium brevicompactum (Pbfds) was performed using TAIL-PCR and RT-PCR followed by complementation tests in Saccharomyces cerevisiae and determination of its expression profile by semi-quantitative RT-PCR. Promoter analysis suggests some binding sites for transcription factors some of which are involved in fungal growth and response to environmental stress. The Pbfds ORF encodes a cytosolic 39.7 kDa protein with a high conservation among Eurotiomycetes and the highest identity (96 %) with Pen. chrysogenum. Homology-based structural modeling suggests that the PbFDS is formed by the arrangement of 15 core helices around a large central cavity where the catalytic reaction takes place. Superimposition of the predicted 3D structure of the enzyme on its ortholog in human reveals the same folding pattern in the counterparts. CONCLUSION: The Pbfds expression may be stimulated in response to the environmental stresses and fungal growth and encodes the PBFDS--a cytosolic enzyme which with a key role in ergosterol and secondary metabolites biosynthesis.